Nitric oxide (NO) bioavailability in the body is important in maintaining several aspects of homeostasis, and its dysfunction contributes to a large variety of diseased states. In endothelial cells, NO is produced by endothelial nitric oxide synthase and can diffuse from the endothelial cells to the smooth muscle cells, where it causes vasodilation via activation of soluble guanylate cyclase (Palmer et al., Nitric-Oxide Release Accounts for the Biological-Activity of Endothelium-Derived Relaxing Factor. Nature 327 (6122), 524 (1987)). In the body, nitric oxide is a modulator of, inter alia, vascular permeability (Yuan et al., New insights into eNOS signaling in microvascular permeability. Am J Physiol Heart Circ Physiol 291 (3), H1029 (2006)), angiogenesis (Murohara et al., Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. The Journal of clinical investigation 101 (11), 2567 (1998)), platelet adhesion and aggregation (Loscalzo, Nitric oxide insufficiency, platelet activation, and arterial thrombosis. Circulation research 88 (8), 756 (2001)), and leukocyte adhesion (Kubes et al., Nitric oxide: an endogenous modulator of leukocyte adhesion. Proceedings of the National Academy of Sciences of the United States of America 88 (11), 4651 (1991)).
Nitric oxide activity is rapidly diminished in the presence of hemoglobin (Hb). Hemoglobin (Hb) reacts with NO primarily through the dioxygenation reaction (Scheme 1), in which oxygenated Hb (OxyHb) reacts with NO to form Methemoglobin (MetHb, where the heme is oxidized to FeIII) and nitrate (NO3−). Nitric oxide also binds to a ferrous vacant heme (Scheme 2) of deoxygenated Hb (deoxyHb) to form iron nitrosyl Hb (FeIINO-Hb).HbO2+NO→MetHb+NO3−  (1)Hb+NO→FeIINO-Hb   (2)
These reactions occur at nearly diffusion-limited rates: 5-8×107 M−1s−1 for the dioxygenation reaction, and 3×107 M−1s−1 for the NO binding reaction to deoxyHb (Huang et al., Nitric Oxide Red Blood Cell Membrane Permeability at high and low Oxygen Tension. Nitric Oxide 16, 209 (2007)). The production of nitrate from the dioxygenation reaction is a dead end with respect to NO bioactivity. In addition, any NO that is slowly released from iron nitrosyl Hb is likely to be scavenged by OxyHb, thereby destroying its activity.
In the vascular tissues, nitric oxide is made in a compartment adjacent to the blood, where there is 10 mM Hb (in heme). This presents a paradox as to how NO can function without being scavenged by the Hb (Lancaster, Simulation of the Diffusion and Reaction of Endogenously Produced Nitric-Oxide. Proc. Natl. Acad. Sci. USA 91 (17), 8137 (1994)). Based on kinetic calculations in normal physiology, it is thought that endothelial-derived NO is not scavenged to the extent predicted because red blood cell (RBC) encapsulated Hb in the blood reacts with NO much more slowly than does cell-free Hb (Vaughn et al., Erythrocytes possess an intrinsic barrier to nitric oxide consumption. J. Biol. Chem. 275 (4), 2342 (2000)).
Without wishing to be bound by theory, three mechanisms are thought to contribute to reduced NO scavenging by RBCs. First, the rate of the reaction is largely limited by external diffusion of NO to the RBC. Second, NO diffusion is partially blocked by a physical barrier across the RBC membrane. Third, RBC encapsulated Hb is efficiently compartmentalized in the lumen; it does not extravasate into the endothelium and interstitium (Kim-Shapiro et al., Unraveling the Reactions of Nitric Oxide, Nitrite, and Hemoglobin in Physiology and Therapeutics. Arterioscler Thromb Vasc Biol 26, 697 (2006)).
All three of these mechanisms break down during hemolysis, in which destruction of the RBCs results in release of Hb into the blood plasma, where it can scavenge NO. Supporting this notion, the increased ability of cell-free Hb to scavenge NO has been attributed to the hypertension, increased systemic and pulmonary vascular resistance, and morbidity and mortality associated with administration of hemoglobin-based oxygen carriers (HBOCs or “blood substitutes”) (Doherty et al., Rate of reaction with nitric oxide determines the hypertensive effect of cell-free hemoglobin. Nature Biotechnology 16 (7), 672 (1998)).
There is also a host of animal and human data supporting the theory that NO scavenging by cell-free Hb due to intravascular hemolysis contributes to disease. For example, the importance of intravascular hemolysis on NO bioavailability in diseased states including hemolytic anemias such as sickle cell disease and paroxysmal nocturnal hemoglobinuria (PNH), thalassemia intermedia, malaria, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome and cardiopulmonary bypass has been elucidated (Gladwin, M. T., Unraveling the hemolytic subphenotype of sickle cell disease. Blood 106 (9), 2925 (2005); Minneci et al., Hemolysis-associated endothelial dysfunction mediated by accelerated NO inactivation by decompartmentalized oxyhemoglobin. J. Clin. Invest. 115, 3409 (2005); Rother et al., The clinical sequelae of intravascular hemolysis and extracellular plasma hemoglobin—A novel mechanism of human disease. Jama-J Am Med Assoc 293 (13), 1653 (2005)).
It has been shown that hemolysis in cardiopulmonary bypass surgery leads to renal tube injury and other complications (Tanaka et al., Administration of Haptoglobin during Cardiopulmonary bypass surgery. Trans. Am. Soc. Artif. Intern. Organs 37, M482 (1991)). Minneci et al. demonstrated that intravascular hemolysis leads to vasoconstriction and impairs renal function in a canine model (Hemolysis-associated endothelial dysfunction mediated by accelerated NO inactivation by decompartmentalized oxyhemoglobin. J. Clin. Invest. 115 (12), 3409 (2005)).
Reiter et al. found that responsiveness to NO administration was blunted by 80% in patients with sickle cell anemia who had plasma heme concentrations greater than or equal to 6 μM (Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease. Nat. Med. 8 (12), 1383 (2002)). The hemolysis in sickle cell disease is generally lower than that of other conditions, with an average of 4.2±1.1 μM during steady state, compared to 0.2±0.1 μM for control normal volunteers. However, hemolysis increases several fold during sickle cell crisis (Naumann et al., Plasma hemoglobin and hemoglobin fractions in sickle cell crisis. Am. J. Clin. Pathol. 56, 137 (1971); Ballas et al., Hyperhemolysis during the evolution of uncomplicated acute painful episodes in patients with sickle cell anemia. Transfusion 46 (1), 105 (2006)).
We have conducted calculations demonstrating that only 1 μM cell-free Hb significantly reduces NO bioavailability, even in the background of the 10 mM or so Hb (in heme) found in whole blood (Jeffers et al., Computation of plasma hemoglobin nitric oxide scavenging in hemolytic anemias. Free Radic. Biol. Med. 41 (10), 1557 (2006)). Thus, pathology associated with low NO bioavailability is an important contributor to pathology in conditions involving hemolysis.
Reiter et al. showed that NO inhalation therapy can result in conversion of plasma OxyHb to MetHb in patients with sickle cell disease, thereby reducing the enhanced NO scavenging of the plasma Hb (Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease. Nat. Med. 8 (12), 1383 (2002)). Similarly, in the canine model, Minneci et al. showed that NO inhalation following hemolysis resulted in restoration of NO responsiveness to NO donors and attenuation of the hemolysis-associated vasoconstriction (Hemolysis-associated endothelial dysfunction mediated by accelerated NO inactivation by decompartmentalized oxyhemoglobin. J. Clin. Invest. 115 (12), 3409 (2005)). These results support the approach of oxidizing the cell-free Hb to diminish NO scavenging. Indeed, NO inhalation therapy in sickle cell disease and other hemolytic conditions has been gaining increased attention.
Although use of NO inhalation therapy holds promise for treatment of hemolytic conditions, its use is not practical in a variety of settings, particularly where chronic treatment is desired. NO inhalation therapy is expensive, and compliance in its use with portable gas cylinders is not likely to be great. In addition, formation of MetHb as an end-product during NO therapy may not be ideal due to potential oxidative damage (Alayash, Oxygen therapeutics: Can we tame haemoglobin? Nat Rev Drug Discov 3 (2), 152 (2004); Motterlini et al., Oxidative-Stress Response in Vascular Endothelial-Cells Exposed to Acellular Hemoglobin-Solutions. Am. J Physiol.-Heart Circul. Physiol. 38 (2), H648 (1995); Balla et al., Endothelial-Cell Heme Uptake from Heme-Proteins—Induction of Sensitization and Desensitization to Oxidant Damage. Proc. Natl. Acad. Sci. USA 90 (20), 9285 (1993)).
Therefore, new approaches are needed in the treatment of conditions associated with hemolysis.